In vitro effects of pyrethroids on rat brain and liver ATPase activities

In vitro sensitivity of rat brain and liver ATPases to pyrethroid insecticides, belonging to three categories based on the structural configuration, was studied. Rat brain and liver P2 fractions were prepared by the conventional centrifugation method, and rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Na+, K+-ATPase and oligomycin-sensitive and -insensitive Mg2+- ATPases were determined in brain P2 fraction, whereas in liver P2 fraction only oligomycin-sensitive and -insensitive Mg2+-ATPase activities were determined. [3H]Ouabain binding studies were carried with rat brain synaptosomes. Most of the pyrethroid compounds tested inhibited brain and liver oligomycin-sensitive Mg2+-ATPases at micromolar concentrations. Type II compounds were more effective as compared to Type I compounds. Oligomycin-insensitive Mg2+-ATPase was not affected by any of the compounds tested except deltamethrin, which showed significant effect on liver enzyme. Na+, K+-ATPase of brain was less sensitive to these pyrethroids as compared to oligomycin-sensitive Mg2+-ATPase. [3H]Ouabain binding to rat brain synaptosomes was not affected significantly by these pyrethroid insecticides. These results suggest that inhibition of oligomycin-sensitive Mg2+-ATPase may be involved in the toxicity of pyrethroid compounds.     

Effect of captopril on Na-K ATPase and Mg ATPase activities of erythrocyte ghost membranes

Captopril has a known hypotensive action derived from the inhibition of the converting enzyme (Kininase II). To investigate whether other mechanism may contribute to vasodilation, Na-K ATPase and Mg ATPase activities on ghost red blood cells membranes were examined in the presence of the drug. The results show a stimulation of Mg ATPase activity and an inhibition of Na-K ATPase activity in a concentration dependent manner. The latter action does not seem to be related to a vasodilator effect.     

多药耐药逆转剂与血脑屏障上P-糖蛋白ATP酶活性间的相互作用(英文)

目的:进一步探讨P-糖蛋白(P-gp)与其多种耐药逆转剂间ATP依赖性相互作用的机制.方法:从牛脑灰质中分离得到微血管内皮细胞(BCEC),制成细胞膜,定磷法测定BCEC膜上P-gp ATPase活性.结果:维拉帕米(Ver)、长春新碱(VCR)、阿霉素(Dox)、粉防己碱(Tet)、蝙蝠葛碱(DRC)、小檗胺(BBM)以及蝙蝠葛苏林碱(DRS)增加基础P-gpATPase活性,其Km值分别约为17、5.9、41、2.3、11、23和22μmol/L.小檗碱(BBR)仅有轻微的激活作用,延胡索乙素(dl-THP)和左旋四氢巴马汀(l-THP)不改变基础P-gp ATPase活性.环孢素A(CsA)抑制基础P-gp ATPase活性;竞争性抑制Ver或VCR激活的P-gp ATPase活性;非竞争性抑制Dox或Tet激活的P-gp ATPase活性.Dox非竞争性抑制Tet-激活的P-gp ATPase活性.结论:各种多药耐药逆转剂与P-gp相互作用的机制及其对P-gp ATPase活性的影响各不相同.CsA、Ver和VCR在血脑屏障P-gp上的结合部位可能是重叠的或者是有相互联系的,而CsA、Dox和Tet与P-gp的结合是相互独立的,并存在各自不同的结合部位.     

多药耐药逆转剂与血脑屏蔽上P—糖蛋白ATP酶活性间的相互作用

目的：进一步探讨P－糖蛋白（P-gp)与其多种耐药逆转剂量ATP依赖性相互作用的机制。方法：从牛脑灰质中分离得到微血管内皮细胞（BCEC）,制成细胞膜,定磷法测定BCEC膜上P-gp ATPas活性,结果：维拉帕米（Ver),长春新碱（VCR）,阿霉素（Dox),粉防己碱（Tet),蝙蝠葛碱（DRC）,小檗胺（BBM）以及蝙蝠葛苏林碱（DRS）增加基础P-gp ATPas活性,其Km值分别约为17,5．9,41,2．3,11,23和22μmol/L,小檗碱（BBR）仅有轻微的激活作用,延胡索乙素（dl-THP)和左旋四氢巴马汀（l-THP)不改变基础P-gp ATPas活性。环孢素A（CsA)抑制基因P-gp ATPas活性;竞争性抑制Ver或VCR激活的P-gp ATPas活性;非竞争性抑制Dox或Tet激活的P-gp ATPas活性。Dox非竞争性抑制Tet-激活的P-gp ATPas活性。结论：各种多药耐药逆转剂与P-gp 相互作用的机制及其对P-gp ATPas活性的影响各不相同,CsA,Ver和VCR在血脑屏蔽P-gp 上的结合部位可能是重叠的或者是有相互联系的,而CsA,Dox和Tet与P－gp的结合是相互独立的,并存在各自不同的结合部位。     

Inhibitory effects of pomelo on the metabolism of tacrolimus and the activities of CYP3A4 and P-glycoprotein.

We recently reported a case of increase in the blood level of tacrolimus following intake of pomelo in a renal transplant recipient. To clarify the mechanism of this increase in the blood level of tacrolimus, we investigated the effect of pomelo juice extract on the activities of CYP3A4 and P-glycoprotein, in comparison with that of extract of grapefruit juice (GFJ). The 10% ethyl acetate extracts of the juice of three pomelos of different origins (Banpeiyu, pomelo I; Hirado Buntan, pomelo II; and Tosa Buntan, pomelo III) and GFJ significantly inhibited 6beta-hydroxylation of testosterone in human liver microsomes by 76.4, 67.2, 37.5, and 83.9%, respectively. The extract of pomelo I was as potent as that of GFJ. The metabolism of tacrolimus itself was also inhibited by the extract of pomelo I, as well as that of GFJ. Furthermore, the inhibition of both 6beta-hydroxylation of testosterone and metabolism of tacrolimus by pomelo I and GFJ was preincubation time-dependent. On the other hand, the extract of pomelo I had little effect on the transcellular transport of tacrolimus or [(3)H]digoxin across a monolayer of LLC-GA5-COL150 cells (a porcine kidney epithelial cell line, LLC-PK1, transfected with human MDR1 cDNA and overexpressing human P-glycoprotein). In conclusion, pomelo constituents inhibit the activity of CYP3A4 and may thereby produce an increase in the blood level of tacrolimus.     

Structure-activity relationship studies of propafenone analogs based on P-glycoprotein ATPase activity measurements.

Propafenone analogs (PAs) were previously identified as potent inhibitors of P-glycoprotein (Pgp)-mediated toxin efflux. For this as well as other classes of Pgp inhibitors, lipophilicity as well as hydrogen bond acceptor strength are important determinants of biological activity. The question as to whether a direct interaction between PA-type modulators and Pgp takes place was addressed by means of Pgp ATPase measurements and transport studies. Propafenone-type modulators stimulated ATPase activity up to 2-fold over basal activity in a concentration-dependent biphasic manner. Within a series of structural homologs, Ka values of ATPase stimulation strongly correlated with lipophilicity. Analogs containing a quaternary nitrogen stimulated Pgp ATPase activity with lesser efficacy, while Ka values were somewhat higher when compared to corresponding tertiary analogs. Transport studies performed in inside-out plasma membrane (I/O) vesicles demonstrated that analogs containing a tertiary nitrogen rapidly associated with the biomembrane. Quaternary analogs, which are restricted by a permanent positive charge in transiting the plasma membrane by diffusion, accumulated in Pgp containing I/O vesicles in an ATP-dependent and cyclosporin A-inhibitable manner, which identified them as Pgp substrates. Identical structure-activity relationships were found in either Pgp ATPase stimulation experiments in I/O vesicles or in toxin efflux inhibition studies using intact cells. Therefore, differences in membrane transit are not responsible for the observed structure-activity relationships.     

Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein

1. P-glycoprotein, a 170–180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3′-keto-Bmt]-[Val^*]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein.
2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay.
3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity.
4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833.
5. The binding of 8-azido-[α-³²P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein.
6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.

     

Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence on ATPase activity and membrane fluidity

Efflux pump (e.g., P-gp, MRP1, and BCRP) inhibition has been recognized as a strategy to overcome multi-drug resistance and improve drug bioavailability. Besides small-molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and Vitamin E TPGS (TPGS 1000) are known to modulate efflux pump activity. Competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase have been proposed as possible mechanisms. Focusing on TPGS 1000, the aim of our study was to unravel the inhibitory mechanism by comparing the results of inhibition experiments in a Caco-2 transport assay with data from electron spin resonance (ESR) and from ATPase activity studies. ESR results, on Caco-2 cells using 5-doxyl stearic acid (5-SA) as a spin probe, ruled out cell membrane fluidization as a major contributor; change of membrane fluidity was only observed at surfactant concentrations 100 times higher than those needed to achieve full efflux inhibition. Concurrently, TPGS 1000 inhibited substrate induced ATPase activity without inducing significant ATPase activity on its own. By investigating TPGS analogues that varied by their PEG chain length, and/or possessed a modified hydrophobic core, transport studies revealed that modulation of ATPase activity correlated with inhibitory potential for P-gp mediated efflux. Hence, these results indicate that ATPase inhibition is an essential factor in the inhibitory mechanism of TPGS 1000 on cellular efflux pumps.     

Effects of adrenochrome on rat heart sarcolemmal ATPase activities

Effects of adrenochrome on rat heart sarcolemmal ATPase, calcium binding and adenylate cyclase activities were studied _vitro. Adrenochrome (1–100 μg/ml) did not affect calcium binding activity. Significant decreases in adenylate cyclase and Ca²⁺ ATPase activities were seen only at a concentration of 100 μg/ml adrenochrome. Adrenochrome depressed Na⁺?K⁺ ATPase activity in a dose-dependent manner. The inhibitory effect on Na⁺?K⁺ ATPase of adrenochrome was also observed in sarcolemmal membranes washed with a buffer after treatment with adrenochrome. The percentage inhibition of the enzyme activity was independent over a wide range of pH (6.6–7.8) and concentrations of NaCl/KCl (40/4–100/10,mM/mM). A study on the combined effects of adrenochrome and either ouabain or CaCl₂ showed that, unlike ouabain, calcium produced additive inhibition with adrenochrome. Depression of Na⁺?K⁺ ATPase activity was also observed in sarcolemma isolated from the heart perfused with adrenochrome. Since adrenochrome has been demonstrated to produce myocardial cell damage and contractile failure in the perfused heart, the present experiments provide a possible explanation for the genesis of adrenochrome-induced cardiotoxicity.     

P-glycoprotein ATPase activating effect of opioid analgesics and their P-glycoprotein-dependent antinociception in mice

It is well known that opioid analgesics exert central antinociceptive actions. However, in vivo and in vitro studies have shown that some opioid analgesics given systemically have limited access to the central nervous system because of the blood-brain barrier (BBB). P-glycoprotein (P-gp), an ATP-dependent drug efflux transporter, is one component of the BBB. In this report, we assessed the antinociceptive effect of morphine, fentanyl, and meperidine in P-gp deficient (mdr1a KO) mice, and compared these effects with those in wild type (WT) mice. The antinociceptive effects of morphine and fentanyl in mdr1a KO mice were significantly greater than those in WT mice. However, there was no clear difference in the antinociceptive effects of meperidine in the two genotypes. In addition, we determined the effect of opioid analgesics on P-gp ATPase activity, which is requisite for drug transport, using mouse brain capillary endothelial cells. In our observations, morphine and fentanyl, but not meperidine, significantly increased P-gp ATPase activity, and the drugs' concentration-response curves were bell-shaped, reaching a peak at a concentration of 1 muM. These results suggest that P-gp ATPase activity may be, at least in part, involved in the antinociceptive potencies of those opioid analgesics that are substrates for P-gp.     

Biomarkers of endothelial cell activation: candidate markers for drug-induced vasculitis in patients or drug-induced vascular injury in animals

There is a pressing need for vascular biomarkers for studies of drug-induced vasculitis in patients and drug-induced vascular injury (DIVI) in animals. We previously reviewed a variety of candidate biomarkers of endothelial cell (EC) activation (Zhang et al., 2010). Now we update information on EC activation biomarkers from animal data on DIVI and clinical data of vasculitic patients, particularly patients with primary antineutrophil cytoplasmic autoantibody (ANCA)-associated small vessel vasculitis (primary AAVs), including Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and necrotizing crescentic glomerulonephritis. Drug-associated ANCA-positive small vessel vasculitis (drug-AAVs) can closely resemble primary AAVs, suggesting the large overlap between primary idiopathic systemic vasculitis and drug-induced vasculitis. AAVs in patients and DIVI in animals vary considerably; however, there is close resemblance between AAVs and DIVI in some respects: (1) the immunopathogenetic mechanisms (activation of primed neutrophils, ECs and T cells by ANCA in patients and activation of ECs, mast cells, and macrophages by drugs in animals); (2) the morphologic changes (fibrinoid necrosis of the vessel wall and neutrophilic infiltration); (3) the preferable sites (small arteries, arterioles, capillaries and venules); and (4) elevation of vascular biomarkers suggestive of an endothelial origin. The present review discusses soluble and cell component biomarkers and provides a rationale for the potential utility of EC activation biomarkers in nonclinical and clinical studies during new drug development. Further investigation, however, is needed to assess their potential utility.     

Effects of the stilbene derivatives SITS and DIDS on intestinal ATPase activities

4-Acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) effects have been tested both in the basolateral membranes (BLMs) of jejunum enterocytes and in the same intestinal tract, everted and incubated "in vitro". Total and (Na,K)-ATPase activities of BLMs are inhibited in a similar way by the two disulfonic stilbenes as well as the fluid transintestinal transport in the everted intestine; on the contrary cell Na and K are unaffected. SITS and DIDS inhibition of (Na,K)-ATPase seems to take place at the cytoplasmic side of the BLM.